Trypanosoma brucei is the unicellular parasitic protozoa, which causes a fatal disease, African sleeping sickness in human beings and a similar disease in domestic animals. The parasite possesses a single mitochondrion with a concatenated network of mitochondrial DNA known as kinetoplast. In spite of this complex structure, mitochondrial DNA in this protozoal parasite encodes only a few proteins. Thus, similar to the biology of other eukaryotes, the majority of the mitochondrial proteins in T. brucei need to be imported to mitochondria after their synthesis on the cytosolic ribosomes. Our long-term goal is to understand the mitochondrial biogenesis, particularly the mechanistic details of mitochondrial protein import in T. brucei. Many nuclear encoded mitochondrial proteins are crucial for the survival of the mammalian bloodstream forms and many more are needed for the insect or the procyclic form of T. brucei. However, mitochondrial protein import mechanisms are least understood in this group of earliest eukaryotes. In spite of the conservation observed in mitochondrial protein translocases (Toms and Tims) from fungi to mammals, most homologs for these proteins have remained undetectable in trypanosomatid genome databases. In our laboratory, we have recently established an in vitro system for protein import into T. brucei mitochondria. We also have identified and characterized an ortholog of Tim17 from T. brucei. We found that T. brucei Tim17 (TbTim17) is critical for the survival of the procyclic form. It is needed for mitochondrial biogenesis, cell cycle progression and also for cellular morphogenesis. Based on our preliminary results, we hypothesize that TbTim17 plays a central role in mitochondrial biogenesis, which is crucial for many cellular functions in T. brucei. The specific aims for this project are: (1) to evaluate the role of TbTim17 in the import of key proteins into the mitochondria of the bloodstream and procyclic forms of T. brucei;(2) to identify and characterize mitochondrial proteins associated with TbTim17;and (3) to analyze the structure-function relationship of the TbTim17 for understanding the mechanism of its action. We will use RNAi strategies and our rigorously characterized in vitro protein import system to evaluate the role of TbTim17 in mitochondrial protein import. We will isolate a TbTim17-containing protein complex and identify and characterize trypanosome specific proteins associated with TbTim17. Furthermore, we will perform various mutagenesis analyses to evaluate the structural domains of TbTim17 critical for its function and protein-protein interactions. By accomplishing specific aims 1-3, we will elucidate mitochondrial protein import mechanisms in T. brucei and also identify unique and essential protein importer molecules that could be exploited as the target for rational chemotherapeutic intervention.